In addition to the essential structural and regulatory genes, human immunodeficiency virus type 1 has several accessory genes which are well conserved and appear to be necessary in vivo, yet are dispensable for replication in vitro. The essential functions responsible for conservation of these genes in vivo remain uncertain. We have found that one, vpr, is critical for productive HIV-1 infection in primary macrophages. Infection of primary lymphocytes or transformed cells, however, is not dependent on vpr, consistent with many previous reports. The mechanism which underlies its effect in primary macrophage infection is unclear. Our hypothesis is that vpr plays a critical role in HIV-1 infection and replication in primary macrophages, and that its function in macrophage infection may be related to its role in vivo. This First Award application proposes to build on our in vitro model for macrophage infection, our biologically active cloned macrophage-tropic HIV-1 isolate, and preliminary observations on HIV-l accessory gene mutants in these cells. The goal of this study is to better understand the function of vpr in HIV-l infection in primary macrophages using three specific strategies. First, we will define the points in the replication cycle in primary macrophages at which vpr augments productive infection. This will involve a detailed analysis of the viral life cycle in macrophages comparing our molecularly cloned wild-type and vpr-mutant macrophage-tropic viruses. Second, we will do mutational analysis of vpr to determine structure-function relationships in its mechanism of action in macrophage infection. To do this we will employ both biologically active viruses carrying mutant genes and a model utilizing single-cycle infections with a reporter gene. Third, we will determine the role of vpr in infection of a variety of macrophage models relevant to infection and pathogenesis. We will extend our biological studies to macrophages cultured under different conditions in vitro, and to primary macrophages derived from several in vivo tissue sources. Because infected macrophages are believed to play a pivotal role in vivo and vpr appears to have a significant effect in infection of primary macrophages but not other cell types, it is critical to explore its function in a primary macrophage model. It is our expectation that these studies will provide new insights into the mechanism of action of a conserved gene whose function remains to be clarified.